RANKL, also known as OPGL/ODF/TRANCE, is a member of the tumor necrosis factor (TNF) superfamily that plays an important role in osteoclast differentiation, function and survival. RANKL exerts these effects by binding to its receptor RANK, which is expressed on osteoclast precursors and mature osteoclasts. RANKL has been shown to activate six major signaling pathways: NF-kappa B, JNK, ERK, p38, NFATd and AKT. RANK belongs to the TNF receptor (TNFR) family and, hence, it transduces intracellular signals by recruiting various adaptor proteins including TNF receptor associated factors (TRAFs) through the specific motifs in the cytoplasmic domain. In the previous years of this grant we have characterized three TRAF-binding motifs that regulate osteoclast formation, function, and/or survival by activating one or more of the six known signaling pathways. Most importantly, however, we have also elucidated a 4-a.a. novel RANK cytoplasmic motif (IVVY535-538) that plays an essential role in osteoclastogenesis. This novel motif plays a crucial role in osteoclastogenesis by committing macrophages to the osteoclast lineage. Moreover, this RANK motif is NOT involved in the activation of the known RANK signaling pathways, indicating that it initiates a novel pathway(s). Based on these data, we hypothesize that, in addition to the known signaling pathways, RANK also activates an unidentified signaling pathway(s) critical for osteoclastogenesis through the novel motif. Thus, in the current application, we propose to identify and characterize the novel signaling pathways by investigating the following specific aims: 1) Investigate the functional relationship between the three TRAF-binding sites and the novel RANK motif; 2) Identify and characterize the downstream signaling protein that interacts with the novel RANK motif; 3) Investigate the role of the novel RANK motif in osteoclastogenesis in vivo by generating and characterizing knockin mice bearing an inactivating mutation in the novel RANK motif. The identification and characterization of the novel RANK signaling pathways will not only provide crucial insight into the molecular mechanism of osteoclastogenesis, but,more importantly, may lead to the development of more potent and specific therapeutics for various bone diseases including postmenopausal osteoporosis, boss loss in rheumatoid arthritis (RA) and tumor-induced osteolysis.